Microinjection method and device

ABSTRACT

An object of the present invention is to provide a method for introducing a physiologically active substance such as a gene into a cell, which introduces a physiologically active substance such as any given gene into any given cell in a view under a microscope, while significantly reducing invasiveness to the cell, and a device used for the above method. The present invention provides a method for introducing a physiologically active substance into a cell, which comprises: allowing a physiologically active substance to attach around a needle having a diameter of 500 nm or less, provided that it is able to be inserted into a cell; and inserting the above-described needle into the cell; and a microinjection device for carrying out the aforementioned method.

TECHNICAL FIELD

The present invention relates to a method for introducing aphysiologically active substance into cells and a microinjection deviceused for the above method.

BACKGROUND ART

Examples of a technique of introducing gene DNA into cultured cells orthe like may include the calcium precipitation method, the lipidtransfer method, the viral vector method, electroporation, the gene gunmethod, and the microinjection method. In the above methods other thanthe microinjection method, DNA is introduced in cells at a certainprobability, and thus it is impossible to introduce DNA into only aspecific cell. On the other hand, the microinjection method has beenproblematic in that since the diameter of the edge of a glass pipette isapproximately 1 μm, cells are easily damaged when such a glass pipetteis inserted into the cell nucleus thereof. In addition, when differentgenes are introduced into multiple cells, the same number of pipettes asthat of genes should be prepared, resulting in complicated preparation.

Japanese Patent Application Laid-Open No. 2003-88383 discloses that inorder to provide a means for collecting biomolecules such as RNA fromliving cells, a needle capable of specifically binding to biomoleculesis inserted into a living cell using a device enabling fine positioncontrol, and that the needle is then removed from the cell. As a needleused herein, a ZnO whisker or a carbon nanotube is used. For example,the surface of a metal oxide whisker is modified with an amino group, sothat the whisker can bind to biomolecules existing in cells and collectthem.

DISCLOSURE OF THE INVENTION

As mentioned above, the conventional electroporation or gene gun is ableto inject a substance into large quantities of cells at a time. However,it has been difficult to inject a substance into a specific cell.Moreover, the conventional microinjection is able to inject a substanceinto a specific cell. However, since a hollow glass capillary has beenused as a needle to be injected, there has been a certain limitregarding reduction in the external diameter thereof. Thus, theseconventional methods have been problematic in that a cell bursts orsuffers fatal damage when a needle is injected therein, or in thatoperations become complicated.

As described in Japanese Patent Application Laid-Open No. 2003-88383, itis possible to collect biomolecules from living cells by performingspecific modification on a metal oxide whisker or a carbon nanotube. Itis also possible to successively record a change in each of the cellsover time. However, a method for successively recording the change insuch a cell over time by actively introducing a gene therein has not yetbeen disclosed. In addition, the aforementioned method has beenproblematic in that the method comprises a complicated step of modifyingthe surface of a needle with a substance that is allowed to specificallybind to biomolecules.

It is an object of the present invention to solve the aforementionedproblems of the prior art techniques. In other words, it is an object ofthe present invention to provide a method for introducing aphysiologically active substance such as a gene into a cell, whichintroduces a physiologically active substance such as any given geneinto any given cell in a view under a microscope, while significantlyreducing invasiveness to the cell, and a device used for the abovemethod.

As a result of intensive studies directed towards achieving theaforementioned object, the present inventors have found that the objectcan be achieved by using a needle having a diameter of 500 nm or less,provided that it is able to be inserted into a cell, and by insertingthe above-described needle into the cell, thereby completing the presentinvention.

Thus, the present invention provides a method for introducing aphysiologically active substance into a cell, which comprises: allowinga physiologically active substance to attach around a needle having adiameter of 500 nm or less, provided that it is able to be inserted intoa cell; and inserting the above-described needle into the cell.

Preferably, a needle having a diameter between 50 and 100 nm, providedthat it is able to be inserted into a cell, is used.

Preferably, a needle having a length of 5 μm or less is used.

Preferably, a needle having a taper form, provided that it is able to beinserted into a cell, is used.

Preferably, a needle composed of a carbon nanotube is used.

Preferably, a needle composed of silicon is used.

Preferably, a needle composed of a metal oxide is used.

Preferably, a needle having a diameter between 50 and 500 nm, providedthat it is able to be inserted into a cell, has electrical conductivity.

Preferably, the physiologically active substance is DNA, RNA, or aprotein.

Preferably, using a needle charged with an electrical charge opposite tothat of a physiologically active substance, the physiologically activesubstance is allowed to electrostatically attach to the above-describedneedle, and the above-described needle is then inserted into a cell.

Preferably, using a needle to which a voltage opposite to the charge ofa physiologically active substance has been applied, the physiologicallyactive substance is allowed to electrically attach to theabove-described needle, and the above-described needle is then insertedinto a cell.

Preferably, after a negatively charged physiologically active substancehas been allowed to electrostatically attach to a needle that ispositively charged, the above-described needle is inserted into a cell,and the needle is then negatively charged, so that the physiologicallyactive substance is allowed to detach from the needle.

Preferably, after a negatively charged physiologically active substancehas been allowed to electrostatically attach to a needle to which apositive voltage has been applied, the above-described needle isinserted into a cell, and a negative voltage is then applied to theneedle, so that the physiologically active substance is allowed todetach from the needle.

Preferably, negative voltages that change over time are applied to theneedle, so that the physiologically active substance is allowed todetach from the needle.

Preferably, the voltages that change over time are multiple pulsevoltages.

Preferably, the needle to which a voltage opposite to the charge of aphysiologically active substance is applied, is controlled in terms ofvoltage value and the time required for application of the voltage.

Preferably, the method of the present invention comprises the followingsteps:

(1) a step of positively charging a needle;

(2) a step of immersing the needle in a solution comprising a negativelycharged physiologically active substance, so that the physiologicallyactive substance is allowed to attach around the needle;

(3) a step of inserting the needle into a target site in a cell, andthen applying a negative voltage to the needle, so that thephysiologically active substance is allowed to detach from the needle;

(4) a step of removing the needle from the cell; and

(5) a step of repeating the above-described steps (1) to (4), so as tointroduce at least one desired, identical or different, physiologicallyactive substance into each of multiple cells.

In another aspect, the present invention provides a microinjectiondevice, which comprises: a needle having a diameter between 50 and 500nm, provided that it is able to be inserted into a cell; a driving meansfor controlling the movement of the above-described needle that enablesinsertion of the above-described needle into the cell and the removaltherefrom; and a voltage-applying means for applying a voltage tomaintain a physiologically active substance on the surface of theabove-described needle or to remove it from the above surface, whereinthe above-described needle is inserted into a cell and that thephysiologically active substance is then introduced into the cell.

In another aspect, the present invention provides a microinjectiondevice used for the aforementioned method of the present invention,which comprises: a needle having a diameter between 50 and 500 nm,provided that it is able to be inserted into a cell; a driving means forcontrolling the movement of the above-described needle that enablesinsertion of the above-described needle into the cell and the removaltherefrom; and a voltage-applying means for applying a voltage tomaintain a physiologically active substance on the surface of theabove-described needle or to remove it from the above surface, whereinthe above-described needle is inserted into a cell and that thephysiologically active substance is then introduced into the cell.

Preferably, a microinjection device, which comprises a cell-retainingmeans for retaining a cell at a certain site and a microscope forobserving the cell that is retained in the cell-retaining means, isprovided.

Preferably, a microinjection device, which comprises a vessel forreceiving the physiologically active substance, is provided.

Preferably, the microscope for observing the cell is provided with ameans for maintaining culture environment.

Preferably, the driving means for controlling the movement of theabove-described needle, which is connected to the needle, is apiezoelectric element.

Preferably, by the driving means for controlling the movement of theabove-described needle, the needle is inserted into a cell from thedirection of gravitational force.

Preferably, by the driving means for controlling the movement of theabove-described needle, the needle is descended to a certain height withrespect to the surface of the cell-retaining means.

Preferably, a microinjection device which comprises a washing tank foreliminating the physiologically active substance attached to the surfaceof the above-described needle, is provided.

Preferably, the above-described washing tank is used to perform at leastone selected from sterilized water washing, alkali washing, and acidwashing.

Preferably, the time required for application of a voltage to theabove-described needle is shorter than the time at which theabove-described needle stays in a cell.

Preferably, the above-described cell is contained in a culture solution,in which physiologically active substances are dispersed.

In another aspect, the present invention provides a microinjectiondevice, which comprises: a culture solution, in which physiologicallyactive substances are dispersed; a cell-retaining means for retaining acell at a certain site; a needle having a diameter between 50 and 500nm, provided that it is able to be inserted into the cell; a drivingmeans for controlling the movement of the above-described needle, whichis connected to the needle; and a microscope for observing the cellretained in the cell-retaining means; wherein the above-described needleforms a hole that constitutes a pathway for introducing thephysiologically active substance into the cell.

In another aspect, the present invention provides a method forintroducing a physiologically active substance into a cell, whichcomprises performing microinjection using the aforementionedmicroinjection device of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a summary of the method of the present invention.

FIG. 2 shows a microinjection device constructed on the stage of aninverted microscope.

FIG. 3 is a top view of the microscope stage (the internal view of anincubator).

FIG. 4 shows the positions of the microinjection device and of theneedle used for gene introduction.

FIG. 5 shows the alternate voltage ±5 V at 100 Hz that is used as avoltage to be applied.

FIG. 6 shows a voltage waveform obtained during the period ranging fromthe retention of gene DNA to the release thereof.

FIG. 7 shows the state of cells that are adjacent to each other.

FIG. 8 shows an example of the voltage pattern of the applied voltage.

FIG. 9 shows another example of the voltage pattern of the appliedvoltage.

FIG. 10 shows a schematic view showing a case where gene DNA to beintroduced into a cell is dispersed in a culture solution.

FIG. 11 shows a needle composed of a carbon nanotube, which has adiameter of 50 nm and a length of 3 μm.

FIG. 12 shows a needle produced by narrowing the diameter of acantilever made from silicon by etching, and forming a platinum layer onthe surface thereof.

In the above drawings, 1 represents a cantilever, 2 represents a needle,3 represents a cell, 4 represents a cell nucleus, 5 represents a petridish, 6 represents a cell-retaining means, 7 represents a vessel, 8represents a solution containing a physiologically active substance, 9represents a driving means, 10 represents an electricpotential-controlling means, 11 represents a microinjection device, 12represents an incubator, 13 represents a heater, 14 represents a fan, 15represents an object glass, 16 represents a specimen, 17 represents alight source used for transillumination, 18 represents a vessel, 19represents a washing tank, 20 represents an XY stage, 21 represents aneedle, 22 represents a stacked piezoelectric actuator, 23 represents afixed block, 24 represents a Z-axis stage, 25 represents the bottom of apetri dish, 26 represents a cell, 27 represents a cell nucleus, 28represents gene DNA, and 29 represents a pinhole.

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiments of the present invention will be described in detailbelow.

In the method of the present invention, a physiologically activesubstance is allowed to attach around a needle having a diameter of 500nm or less, and the above needle is then inserted into a cell, so as tointroduce the physiologically active substance into the cell.

The present invention is characterized in that an extremely thin needle(to such an extent that it exceeds optical resolution) is used for geneintroduction. Specifically, a needle having a diameter of 500 nm orless, and particularly preferably having a diameter between 50 and 100nm, can be used, provided that it is able to be inserted into a cell.The needle used in the present invention is preferably a needle, theelectrical properties of which, such as electrification, can easily becontrolled. In the present invention, for example, the surface of theneedle is positively charged, and DNA molecules are allowed to attach tothe surface. Thereafter, the needle is inserted into the cell nucleus,and the surface of the needle is then negatively charged, so as to allowthe DNA molecules to detach from the surface of the needle. Since a thinneedle is used in the present invention, damage given to the cell can bereduced to a minimum, and further, any given DNA can be introduced intoany given target cell.

It has been known that when cell organelle (Golgi body, mitochondrion,and the like) is damaged with a needle, the survival rate of the celldecreases. When a needle having a diameter between 50 and 500 nm isused, provided that it is able to be inserted into a cell, such a needleis small enough with respect to the size of a cell nucleus, and thus ithardly hurts cell organelle other than the cell nucleus. Moreover, byallowing the needle to proceed to the cell from the position directlyabove (from the direction of gravitational force), the needle can beinserted into a site at which the cell nucleus is closest to the cellmembrane. Herein, the probability that the cell organelle exists in avery small space between the cell nucleus and the cell membrane is low.From this viewpoint as well, the cell organelle is hardly damaged, andthus the survival rate of cell can be improved.

In the present invention, only the use of a needle having a surface withconductivity (electrification) is required. Modification of the surfaceof the needle depending on biomolecules is not particularly necessary.

For example, 100 types of DNA solutions and 100 cells are prepared.Thereafter, a needle is immersed in such a DNA solution, and the needleis then inserted into such a cell from the position directly above thecell. Such an operation to immerse a needle in a DNA solution and anoperation to insert the needle into the cell are repeated, so thatdesired individual DNAs can be introduced into different cells, and sothat the cells can individually be transformed. Therefore, according tothe method of the present invention, screening of an agent or exhaustiveanalysis of interaction between biomolecules can be carried out at asingle cell level, differing from the conventional methods wherein suchscreening or analysis is carried out at a well level, using a 96-wellplate or 384-well plate.

The material of the needle used in the present invention is notparticularly limited, as long as it has the aforementioned properties.For example, a carbon nanotube can be used as a needle. Such a carbonnanotube has a cylindrical form obtained by winding a monolayer graphite(graphin), and it is a microcrystal composed of 100% carbon atoms. Inrecent years, nanotechnology has become a focus of attention, and such acarbon nanotube has also received attention in various fields. Examplesof studies regarding the use of a carbon nanotube include thedevelopment of a screen in which a nanotube is used for electron gun, inplace of liquid crystal or plasma display; application of a carbonnanotube to fuel cells and solar cells; and the use as a material forhydrogen storage. A carbon nanotube can be applied to the aforementionedtechniques because it has various types of characteristic propertiessuch as minuteness, the properties as a quantum obtained from itsthree-dimensional structure, and its composition purely consisting ofcarbons, and thus because it has unique properties different from thoseof the conventional materials. In addition, a carbon nanotube purelyconsists of carbons. Thus, differing from carbon black and the like, itcontains almost no impurities. Moreover, a carbon nanotube is alsocharacterized in that it does not change even after it has been exposedto a high temperature during a molding process and/or when it is used.

At present, as a multi wall carbon nanotube, a carbon nanotube having adiameter between approximately 50 and 100 nm and a length of 3 μm ormore is available. It is preferable to use such a carbon nanotube in thepresent invention. If the diameter of a needle is too thin, the amountof a physiologically active substance that can be retained by the needledecreases. In contrast, if the diameter is too thick, the invasivenessto a cell increases. Thus, both cases are unfavorable. Accordingly, inthe present invention, a needle having a diameter of 500 nm or less, andmore preferably a diameter between 50 and 100 nm, is used, provided thatit is able to be inserted into a cell. With regard to the length of aneedle, since the height of a common cultured cell is approximately 5μm, a needle having a length of 5 μm or less can appropriately be used.For example, a needle having a length of approximately 3 μm can be used.

Except for the aforementioned examples, the following needles can alsobe used.

A needle, which is produced by coating a metal oxide whisker shown inthe aforementioned example of prior art technique with gold (Au) orplatinum (Pt) using evaporation or sputtering device or the like, so asto impart electric conductivity to the surface thereof, can be used.Moreover, a needle, which is produced by coating a cantilever made fromsilicon that has frequently been used as a cantilever for an atomicforce microscope with gold (Au) or platinum (Pt) using evaporation orsputtering device or the like, so as to impart electric conductivity tothe surface thereof, can also be used. The needlepoint of such acantilever made from silicon is etched using a device such as IPC or FIBfor acumination, and a conductive membrane is then formed thereon,thereby further reducing the invasiveness to living cells. Furthermore,in the case of a cantilever made from silicon, the needlepoint thereofis converted to a taper form by etching, thereby improving the strengthof the needle. In order to reduce damage given to a cell, it isnecessary for the aforementioned needle to have a diameter between 50and 500 nm, provided that it is able to be inserted into the cell. Inthe case of the aforementioned needle having a taper form also, theneedle has a diameter between 50 and 500 nm, provided that it is able tobe inserted into the cell.

The type of a physiologically active substance that can be introducedinto a cell by the method of the present invention is not particularlylimited. Examples thereof may include nucleic acids such as DNA or RNAand proteins. A preferred example may be a nucleic acid. As such anucleic acid, either DNA or RNA may be used. In addition, examples ofDNA used herein may include genomic DNA or a fragment thereof, cDNA, andsynthetic DNA such as a synthetic oligonucleotide.

In the present invention, a needle having a diameter between 50 and 500nm provided that it is able to be inserted into a cell as mentionedabove (that is, a carbon nanoprobe or a metal oxide whisker having aconductive surface), is attached to the tip of the cantilever of anatomic force microscope (AFM), thereby producing electrical connection.The term “electrical connection” is herein used to mean electricalconnection for positively or negatively controlling the charge of theneedle. This. cantilever works with the image processing of themicroscope and moves between a vessel containing a physiologicallyactive substance of interest and a cell of interest (a cell nucleus orthe like), so that the physiologically active substance of interest canbe introduced into only the cell of interest. In a preferred embodimentof the present invention, the needle is always positioned in thevertical direction, and it controls its needlepoint position at highaccuracy.

The aforementioned movement of the needle can be conducted by a drivingmeans for controlling the movement of the needle. That is to say, thepresent invention provides a microinjection device comprising: a needlehaving a diameter between 50 and 500 nm, provided that it is able to beinserted into a cell; and a driving means for controlling the movementof the above needle. More specifically, the microinjection device of thepresent invention comprises: (a) a cell-retaining means for retaining acell at a certain site; (b) a needle having a diameter between 50 and500 nm, provided that it is able to be inserted into the cell, and adriving means for controlling the movement of the above needle, which isconnected to the above needle; and (c) a microscope for observing thecell retained in the cell-retaining means.

In the present invention, using a needle charged with an electricalcharge opposite to that of a physiologically active substance, thephysiologically active substance may be allowed to electrostatically(electrically) attach to the above needle, and the needle may be theninserted into a cell. When a physiologically active substance having anegative electric charge, such as DNA, is introduced into a cell, theabove physiologically active substance is allowed to electrostatically(electrically) attach to a needle that is positively charged, and theneedle may be then inserted into the cell.

In the present invention, utilizing the electrical polarity of aphysiologically active substance, such a physiologically activesubstance may be allowed to attach to the surface of a needle for acertain period of time, or may be allowed to detach therefrom. That is,needless to say, such a physiologically active substance may be not onlyallowed to electrostatically attach to the surface of a needle, but itmay be also allowed to attach thereon even in a state where a voltage iscontinuously applied to the needle. Thus, it is also possible that thephysiologically active substance may be allowed to detach from thesurface of the needle by reversing (inversing) the polarity of thevoltage applied.

As mentioned above, a method for applying a voltage to a needle is notnecessarily limited to electrostatic action, but it can be selecteddepending on the electrical properties of the physiologically activesubstance or the purpose of use. Thus, the present invention can beapplied to various purposes.

As an example, the method of the present invention comprises thefollowing steps:

(1) a step of positively charging a needle;

(2) a step of immersing the needle in a solution containing a negativelycharged physiologically active substance, so that the physiologicallyactive substance is allowed to attach around the needle;

(3) a step of inserting the needle into a target site in a cell, so asto introduce the physiologically active substance into the cell;

(4) a step of removing the needle from the cell, and then negativelycharging the needle, so as to eliminate the physiologically activesubstance remaining around the needle; and

(5) a step of repeating the above-described steps (1) to (4), so as tointroduce at least one desired, identical or different, physiologicallyactive substance into each of multiple cells.

Hereafter, an example of the embodiments of the present invention willbe described with reference to drawings.

FIG. 1 shows a summary of the method of the present invention. FIG. 1shows with a double-headed arrow that a needle 2 equipped in acantilever 1 that is connected to a driving means 9 moves between theposition directly above a cell 3 and the position directly above avessel 7 comprising a solution 8 containing a physiologically activesubstance. The cell 3 is cultured in a petri dish 5, and the petri dish5 is placed on a cell-retaining means 6.

First, the needle 2 is inserted into the vessel 7 comprising thesolution 8 containing a physiologically active substance, so that thesolution containing the physiologically active substance is allowed toattach to the surface of the needle 2. Such attachment of the abovesolution takes place as a result of the control of the charge of theneedle by an electric potential-controlling means, which is electricallyconnected to the needle 2. Namely, when the physiologically activesubstance is a negatively charged substance such as a nucleic acid, thephysiologically active substance is allowed to efficiently attach to theneedle 2 by positively charging the needle 2 by the electricpotential-controlling means 10 in advance.

Subsequently, the needle 2, to which the physiologically activesubstance has been attached, is lifted up, so that it is removed fromthe vessel 7 comprising the solution 8 containing the physiologicallyactive substance. Thereafter, the needle moves in the horizontaldirection, and it is then disposed at the position directly above thecell of interest 3. The needle 2 disposed at the position directly abovethe cell 3 moves downwards, so that it can be inserted into a cellnucleus 4 in the cell of interest 3. The needle inserted into the cellnucleus 4 then releases the physiologically active substance attached tothe surface thereof to the inside of the cell nucleus 4 under the statusquo. The physiologically active substance can be released by controllingthe charge of the needle by the electric potential-controlling means 10,which is electrically connected to the needle 2. Namely, when thephysiologically active substance is a negatively charged substance suchas a nucleic acid, the needle 2 is negatively charged by the electricpotential-controlling means 10, so that the physiologically activesubstance can efficiently be released from the needle 2. After thephysiologically active substance has been released to the inside of thecell nucleus 4, the needle is removed from the cell. Thereafter, theaforementioned operations are repeated, so as to introduce the desiredphysiologically active substance into the desired cell nucleus. Theaforementioned movements of the needle 2 are all controlled by thedriving means 9.

The present invention further relates to a microinjection device, whichcomprises: a needle having a diameter between 50 and 500 nm, providedthat it is able to be inserted into a cell; a driving means forcontrolling the movement of the above needle that enables insertion theabove needle into the cell and the removal therefrom; and avoltage-applying means for applying a voltage so as to retain aphysiologically active substance on the surface of the above needle orto remove it from the surface thereof, wherein the above needle isinserted into the cell, so as to introduce the physiologically activesubstance into the cell.

The above microinjection device will be described in detail below.

As shown in FIG. 2, the microinjection device is constructed on thestage of an inverted microscope, enabling the observation and/ormeasurement of the process from initiation of gene introduction to thesubsequent course. On the microscope stage, an incubator 12 formaintaining the temperature at 37° C. is constructed, and themicroinjection device is installed in the incubator.

FIG. 3 is a top view of the microscope stage (the internal view of anincubator). The incubator is composed of a metal having excellent heatconductivity (an aluminum alloy or the like). A heater 13 or a fan 14for stirring the internal air is disposed on the internal side of theincubator. The external surface of the incubator is covered with a heatinsulator in order not to release the heat to the external environment.In addition, in order to observe the inside of the incubator with aninverted microscope, some regions on the top and bottom surfaces of theincubator are made from glass, so that a specimen 16 (a cell in a petridish or the like) can be observed with an object glass 15. Moreover, alight from a light source for transillumination can be applied to thespecimen, so that phase difference observation or differentialinterference observation can be conducted.

In order to optimize the pH of a cell culture solution to the cultureenvironment, 5% CO₂ is supplied from the outside of the incubatorthrough a tube, and using a fan, the inside of the incubator isuniformly filled with 5% CO₂.

On the microscope stage formed in the incubator, a petri dish acting asa specimen (a dish, a microplate, or the like), a vessel 18 comprising asolution containing gene DNA, such as a sample cup, a washing tank 19for washing the needle, and the like, are placed on an XY stage 20 thatis operated with a motor or the like.

Accordingly, the specimen 16, the vessel 18, and the washing tank 19 areable to move under a needle used for gene introduction. Thus, as statedabove, the whole area in the specimen can be observed.

A needle 21 used for gene introduction moves only in the Z direction (inthe direction of gravitational force), and thus, it moves up and downwith respect to the target that is positioned under the needle. As shownin FIG. 4, the needle 21 used for gene introduction is disposed, withthe needlepoint thereof directed downwards, with respect to one end faceof a stacked piezoelectric actuator 22 formed by lamination of thinpiezoelectric elements (lead zirconate titanate). The other end face ofthe stacked piezoelectric actuator 22 is disposed on a fixed block 23.When a voltage is applied to the stacked piezoelectric actuator 22, theneedle 21 slightly moves down. A voltage of approximately 100 V realizesa movement of 10 μm, although it depends on the type of a commerciallyavailable stacked piezoelectric actuator. Such amount of displacementcan be controlled with the value of the voltage applied.

Further, the fixed block 23 is mounted on a Z-axis stage 24. With regardto the position of the needle 21 in the Z direction, by a two-stepdriving mechanism in which rough movements are controlled with theZ-axis stage 24 and fine movements are controlled with the stackedpiezoelectric actuator 22, the needle 21 is inserted into the cell 26.For example, the needle 21 is positioned above a petri dish acting asthe specimen 16, it roughly moves to the vicinity of the cell 26 in thepetri dish, and thereafter, when the needle 21 is inserted into the cell26, it finely moves. Since the needle 21 is very easily broken, themovement is terminated immediately before the needlepoint is allowed tocome into contact with the bottom of the specimen 16 such as a petridish (for example, at a height of 1 μm from the bottom).

That is to say, the needle 21 may pass through a cell nucleus 27. Thismicroinjection device can easily be automated by allowing the device torecognize only the step of constantly lifting down the needlepoint to aheight of 1 μm from the bottom. In particular, the control action todetect the surface of a cell membrane and lift down the needlepointseveral μm from the position of the above surface is unnecessary, andthus expensive detection components can be reduced.

On the other hand, when the needle is lifted down to the vessel 18filled with a gene DNA solution or to the washing tank 19, strict heightcontrol is unnecessary, and thus only the rough movement is applied bythe Z-axis stage 24.

The movement of the microinjection device is as described above. Itcomprises the following steps.

(1) Washing of the Needle

The washing tank 19 is positioned under the needle 21 for geneintroduction, and thus the needle 21 is lifted down into the washingtank 19. Washing solution (sterilized water) or the like is stored inthe washing tank 19. An alternate voltage is applied to the needle 21 ina state where the needle is completely immersed in the washing solution.Thus, dusts or gene DNA that has previously been allowed to attach tothe needlepoint are eliminated thereby. For example, as shown in FIG. 5,the alternate voltage ±5 V at 100 Hz is applied as a voltage to beapplied. Thereby, impurities remaining on the surface of the needle areeliminated. Preferably, the washing tank 19 may be subjected toultrasonic washing, or two tanks may be established to wash with agentssuch as acid or alkali and also to wash with sterilized water.

(2) Thereafter, the needle 21 is lifted up from the washing tank 19.During such a step, the needlepoint may be dried by air blowing or thelike.

(3) Subsequently, the vessel 18 containing a gene DNA solution movesunder the needle 21, and the needle is then lifted down therein. Apositive voltage is applied to the surface of the needle in a statewhere the needle is immersed in the solution. For example, a voltage tobe applied is set at 1 V, and the time required for application of sucha voltage is set to be 3 seconds or more. Since the gene DNA has anegative polarity, it is allowed to attach to the surface of the needle.Thereafter, the needlepoint is lifted up, and the specimen 16 isdisposed under the needle. During this step, a voltage may be applied toor may not be applied to the needlepoint.

(4) When the needlepoint is lifted down into a petri dish of thespecimen 16, application of the voltage to the needle 21 is terminated.The needlepoint is lifted down to the vicinity of the cell surface byrough movement, and thereafter, the needle 21 is inserted into the cellnucleus 27 by fine movement.

(5) After the movement of the needle 21 has been terminated, a negativevoltage is applied to the needlepoint, so that the gene DNA on thesurface of the needle is allowed to detach therefrom, thereby beingreleased into the cell (into the cell nucleus). For example, a voltageto be applied is set to be −0.5 V, and the time required for applicationof such a voltage is set to be approximately 1 second. (It is desired toapply a voltage for a time sufficient for the gene DNA attached to theneedle to be detached therefrom.) FIG. 6 shows a voltage waveformobtained during the period ranging from the retention of the gene DNA tothe release thereof.

(6) After completion of the application of the voltage, the needle 21 islifted upwards, and the needle 21 is then washed in the washing tank 19.Thereafter, different gene DNA is allowed to attach to the surface ofthe needle in a vessel containing the different gene DNA, and it is thenreleased into another cell.

Thus, when gene DNA attached to the surface of the needle 21 is releasedinto a cell, a negative voltage is applied to the needle, only when theneedle 21 stays in the cell, so that the gene DNA can be detached fromthe needle by electric repulsion. When a voltage is applied to theneedle in a culture solution, there are concerns about formation ofbubbles as a result of an electrochemical reaction. Thus, in a culturesolution, it is desired to apply a voltage only for a necessary time.Accordingly, it is adequate that a voltage be applied only when theneedle stays in the cell, and that such application of the voltage beterminated during a travel time necessary for the needle to be insertedinto the cell and another travel time necessary for the needle to beremoved from the cell.

By repeating these operations, it becomes possible to introduce eachdifferent gene DNA into cells 26 adjacent to each other, as shown inFIG. 7, and this technique can be used for the analysis of interactionbetween cells and the like. With regard to the conventionalmicroinjection device using a glass tube, the users have manuallycarried out the aspiration and elimination of a gene DNA solution by ahydraulic mechanism or the positioning of the needlepoint to the cell,and thus a certain level of skill has been required. However, with thestructure of the present invention, the cell nucleus 27 is recognizedusing the image processing of the cell, and the needlepoint ispositioned in the center of the cell nucleus 27. The subsequentoperations are easily automated. Thus, the present invention requires nosuch level of skill.

In the aforementioned embodiment, a step of introducing different geneDNA into each cell is described. However, since the needle 21 isextremely thin, causing no substantial damage to the cell, multiple geneDNAs can also be introduced into a single cell.

In addition, the voltage to be applied to the needlepoint is not limitedto the aforementioned example. The voltage patterns shown in FIGS. 8 and9 may also be applied, for example. In FIG. 8, the time required forretaining gene DNA is reduced, and the amount of such gene DNA attachedto the needlepoint can thereby be reduced. That is, the amount of geneDNA to be introduced depends on the amount of gene DNA attached to theneedlepoint. Thus, if the voltage value is decreased and the timerequired for application of the voltage is reduced, the amount of geneDNA attached is reduced. On the contrary, if the opposite operation iscarried out, the amount attached can be increased, and the amountintroduced can thereby also be increased. These operations are effectiveas means for changing the level of gene introduction to cells (makingdifference in the level of gene introduction to cells). In addition, inFIG. 9, when gene DNA is released into the cell nucleus, pulse voltagesare applied to the needlepoints at short intervals (voltages that changeover time), so that the action of removing gene DNA attached to theneedle therefrom can be promoted, and so that almost all amount of geneDNA can be removed. It cannot be said that application of voltages hasno influence upon the cell, and it may become stimulation. Accordingly,the shorter the time required for application of voltages, morepreferable it is. For example, a voltage to be applied is set to be −1V, and 10 pulses of such voltages are applied at 10 Hz.

The stacked piezoelectric actuator 22 used for the fine movement of theneedle can be displaced at a high speed, substantially depending onapplication of a voltage. For example, the natural frequency of astacked piezoelectric actuator 22 having a cross section of 5 mm squareand a length of 20 mm in the bending direction is several kHz. Theneedlepoint moves in response to the voltage pattern at less than theabove frequency (less than the resonant region). Thus, the needle 21 isinserted into the cell 26 at a high speed of several Hz, and the needle21 is then lifted upwards at that cycle. By applying the aforementionedmultiple pulse voltages during the short time from insertion to removal,the time required for the needle 21 to stay in the cell 26 can also bereduced, thereby reducing the invasiveness of the needle to the cell 26.

A case where gene DNA to be introduced into a cell is dispersed in aculture solution, as shown in FIG. 10, will be described below. Theneedle 21 used for gene introduction is moved up and down at a higherspeed (1 cycle frequency consisting of several kHz) by the stackedpiezoelectric actuator 22, so as to form a pinhole 29 on the cellmembrane (cell nucleus 27). As described above, since the needle 21 hasa very small diameter, insertion of such a needle has only littleinfluence upon the cell 26. Moreover, by inserting and removing theneedle 21 at a high speed, damage to the cell 26 can be further reduced.Thereby, a pathway for introducing gene DNA contained in a solution intothe cell 26 (into the cell nucleus 27) is secured. By performing culturein such a state, the gene DNA in the solution can be introduced into thecell. Accordingly, by forming one or more pinholes in each of the cells26 by automatic movement, gene DNA can easily and simply be introducedinto multiple cells.

Preferably, a positive voltage is applied to the needle in a culturesolution, in which gene DNA 28 is dispersed, and the gene DNA isretained on the surface of the needle. Multiple negative pulse voltagesare applied during the time at which the needle is inserted into thecell, so as to release the gene DNA into the cell. The needle moves inthe XY face in the culture solution, so that the gene DNA cansuccessively be introduced into different cells. The operation toimmerse the needle in a vessel so as to retain gene DNA on the surfaceof the needle, the washing operation, and the like, can be omitted, andthereby such gene DNA can be introduced into a large number of cells.Thus, the introduction efficiency is also increased.

Thereafter, the cell, into which a gene has been introduced as mentionedabove, is continuously cultured in an incubator equipped in amicroscope, thereby enabling observing and/or measuring over time, theprocess up to the expression of the gene, or the interaction betweencells.

The present invention will be more specifically described in thefollowing examples. However, the examples are not intended to limit thescope of the present invention.

EXAMPLES Example 1

Using the device shown in FIG. 1, DNA was introduced into nerve cellsthat were cultured in a petri dish for culture.

As nerve cells, PC12. cells (nervous system clone cells isolated fromrat adrenal medulla pheochromocytoma) were used. As a medium, DMEM(Dulbecco's Modified Eagle Medium) containing 10% fetal bovine serum(FBS) was used. The culturing was carried out at 37° C. in 5% CO₂. AsDNA, a recombinant expression vector containing NGF receptor gene wasused, and 1 μg/ml DNA solution was used.

The needle used for the device shown in FIG. 1 composed of the carbonnanotube shown in FIG. 11, which had a diameter of 50 nm and a length of3 μm.

First, the needle was immersed in the DNA solution, so as to allow DNAto attach to the surface thereof. Thereafter, the needle was insertedinto the nucleus of a nerve cell, and then released the DNA therein.After completion of the introduction of the DNA, the nerve cell wascontinuously cultured. Even 3 days later, the nerve cell still survived.

In the case of the needle shown in FIG. 12, the cantilever thereof madefrom silicon was etched for acumination, and a platinum layer was formedon the surface thereof. Using this needle, DNA was introduced into Helacells. As a result, it was found that even 3 days after DNAintroduction, the Hela cells still survived.

On the other hand, as a control, microinjection was carried out using aglass pipette (inside diameter: 300 μm) filled with the same above DNAsolution, instead of using the needle composed of a carbon nanotube witha diameter of 50 nm and a length of 3 μm. After completion of themicroinjection, the nerve cells were continuously cultured. However, thenerve cells died until 3 days later, and no surviving cells existed.

INDUSTRIAL APPLICABILITY

The present invention provides a method for introducing aphysiologically active substance such as any given gene into any givencell that is in a view under a microscope, while significantly reducinginvasiveness to the cell, and a device used for the above method.

1. A method for introducing a physiologically active substance into acell, which comprises: allowing a physiologically active substance toattach around a needle having a diameter of 500 nm or less, providedthat it is able to be inserted into a cell; and inserting said needleinto the cell.
 2. The method of claim 1 wherein a needle having adiameter between 50 and 100 nm, provided that it is able to be insertedinto a cell, is used.
 3. The method of claim 1 wherein a needle having alength of 5 μm or less is used.
 4. The method of claim 1 wherein aneedle having a taper form, provided that it is able to be inserted intoa cell, is used.
 5. The method of claim 1 wherein a needle composed of acarbon nanotube is used.
 6. The method of claim 1 wherein a needlecomposed of silicon is used.
 7. The method of claim 1 wherein a needlecomposed of a metal oxide is used.
 8. The method of claim 1 wherein theneedle having a diameter between 50 and 500 nm, provided that it is ableto be inserted into a cell, has electrical conductivity.
 9. The methodof claim 1 wherein the physiologically active substance is DNA, RNA, ora protein.
 10. The method of claim 1 wherein, using a needle chargedwith an electrical charge opposite to that of a physiologically activesubstance, the physiologically active substance is allowed toelectrostatically attach to said needle, and said needle is theninserted into a cell.
 11. The method of claim 1 wherein, using a needleto which a voltage opposite to the charge of a physiologically activesubstance has been applied, the physiologically active substance isallowed to electrically attach to said needle, and said needle is theninserted into a cell.
 12. The method of claim 10 wherein after anegatively charged physiologically active substance has been allowed toelectrostatically attach to a needle that is positively charged, saidneedle is inserted into a cell, and the needle is then negativelycharged, so that the physiologically active substance is allowed todetach from the needle.
 13. The method of claim 10 wherein after anegatively charged physiologically active substance has been allowed to.electrostatically attach to a needle to which a positive voltage hasbeen applied, said needle is inserted into a cell, and a negativevoltage is then applied to the needle, so that the physiologicallyactive substance is allowed to detach from the needle.
 14. The method ofclaim 10 wherein negative voltages that change over time are applied tothe needle, so that the physiologically active substance is allowed todetach from the needle.
 15. The method of claim 14 wherein the voltagesthat change over time are multiple pulse voltages.
 16. The method ofclaim 11 wherein the needle to which a voltage opposite to the charge ofa physiologically active substance is applied, is controlled in terms ofvoltage value and the time required for application of the voltage. 17.The method of claim 1 which comprises the following steps: (1) a step ofpositively charging a needle; (2) a step of immersing the needle in asolution comprising a negatively charged physiologically activesubstance, so that the physiologically active substance is allowed toattach around the needle; (3) a step of inserting the needle into atarget site in a cell, and then applying a negative voltage to theneedle, so that the physiologically active substance is allowed todetach from the needle; (4) a step of removing the needle from the cell;and (5) a step of repeating the above-described steps (1) to (4), so asto introduce at least one desired, identical or different,physiologically active substance into each of multiple cells.
 18. Amicroinjection device, which comprises: a needle having a diameterbetween 50 and 500 nm, provided that it is able to be inserted into acell; a driving means for controlling the movement of said needle thatenables insertion of said needle into the cell and the removaltherefrom; and a voltage-applying means for applying a voltage tomaintain a physiologically active substance on the surface of saidneedle or to remove it from said surface, wherein said needle isinserted into a cell and that the physiologically active substance isthen introduced into the cell.
 19. (canceled)
 20. The microinjectiondevice of claim 18 which comprises a cell-retaining means for retaininga cell at a certain site and a microscope for observing the cell that isretained in the cell-retaining means.
 21. The microinjection device ofclaim 18 which comprises a vessel for receiving the physiologicallyactive substance.
 22. The microinjection device of claim 20 wherein themicroscope for observing the cell is provided with a means formaintaining culture environment.
 23. The microinjection device of claim18 wherein the driving means for controlling the movement of the needle,which is connected to said needle, is a piezoelectric element.
 24. Themicroinjection device of claim 18 wherein, by the driving means forcontrolling the movement of the needle, said needle is inserted into acell from the direction of gravitational force.
 25. The microinjectiondevice of claim 18 wherein, by the driving means for controlling themovement of the needle, said needle is descended to a certain heightwith respect to the surface of the cell-retaining means.
 26. Themicroinjection device of claim 18 which comprises a washing tank foreliminating the physiologically active substance attached to the surfaceof the said needle.
 27. The microinjection device of claim 26 whereinsaid washing tank is used to perform at least one selected fromsterilized water washing, alkali washing, and acid washing.
 28. Themicroinjection device of claim 18 wherein the time required forapplication of a voltage to said needle is shorter than the time atwhich said needle stays in a cell.
 29. The microinjection device ofclaim 18 wherein said cell is contained in a culture solution, in whichphysiologically active substances are dispersed.
 30. A microinjectiondevice, which comprises: a culture solution, in which physiologicallyactive substances are dispersed; a cell-retaining means for retaining acell at a certain site; a needle having a diameter between 50 and 500nm, provided that it is able to be inserted into the cell; a drivingmeans for controlling the movement of said needle, which is connected tothe needle; and a microscope for observing the cell retained in thecell-retaining means; wherein said needle forms a hole that constitutesa pathway for introducing the physiologically active substance into thecell.
 31. A method for introducing a physiologically active substanceinto a cell, which comprises performing microinjection using themicroinjection device of claim 18.